Functional characterization of CitM, the Mg2+-citrate transporter

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The CitM transporter from Bacillus subtilis is known to transport citrate complexed with Mg2+.
• Understanding its function and substrate specificity is crucial for microbial metabolism studies.

Purpose of the Study:

• To functionally express and characterize the Bacillus subtilis CitM transporter in E. coli.
• To investigate the substrate specificity and ion dependency of CitM-mediated citrate transport.
• To elucidate the mechanism of citrate transport, including proton coupling.

Main Methods:

• Functional expression of CitM in E. coli DH5a.
• Characterization of citrate uptake kinetics in the presence of various metal ions.
• Assessment of transport inhibition by other dicarboxylic and tricarboxylic acids.
• Investigation of the effect of pH and ionophores on citrate transport.

Main Results:

• CitM exhibited a Km for citrate of 274 mM in the presence of saturating Mg2+.
• CitM demonstrated high substrate specificity for citrate, with minimal inhibition by other organic acids.
• The transporter accepted citrate complexes with various metal ions, with a preference order: Mg2+ > Mn2+ > Ba2+ > Ni2+ > Co2+ > Ca2+.
• Citrate transport was proton-coupled, inhibited at alkaline pH (>7.5) and by proton gradient uncouplers, suggesting electroneutral transport.

Conclusions:

• The cloned CitM transporter from B. subtilis expressed in E. coli retains properties similar to its native form.
• The findings support a transport model involving one proton coupled to the uptake of one (Mg2+-citrate)1- complex.
• CitM's ability to transport citrate with different metal ions offers insights into microbial nutrient transport mechanisms.